Br. J. Cancer Br. J. Cancer
'PI
1065-1069 (1992), 66, 1065-1069 (1992), 66,
Macmillan Press Ltd., 1992 1992
Distinction of two different classes of small-cell lung cancer cell lines by enzymatically inactive neuron-specific enolase T.A.W. Splinter', C.F. Verkoelen', M. Vlastuin', T.C. Kok', G. Rijksen2, K.G. Haglid3, F. Boomsma4 & A. van de Gaast' 'Department of Medical Oncology, University Hospital, Dijkzigt, 3015 GD Rotterdam, The Netherlands; 2Department of Haematology, Lab. of Enzymology, University Hospital, 3584 CX Utrecht, The Netherlands; 3Department of Histology, Institute of Neurobiology, University of Goteborg, S-40033 Goteborg, Sweden and 4the Department of Internal Medicine I, University Hospital Dijkzigt 3015 Rotterdam, The Netherlands. Summary Neuron specific enolase (NSE) is widely used
as a neuro-endoctrine marker. However the presence non-neuroendocrine tissues has raised questions on the specificity of NSE. We have investigated NSE immunoreactivity (NSA-ag), y-enolase activity and total enolase activity in small cell lung cancer (SCLC) cell lines. During well-controlled exponential growth comparison of NSE-ag content and y-enolase activity with the doubling-time (Td) and NSE-ag content with y-enolase and total enolase activity led to a clear distinction of two types of cell line: variant cell lines plus part of the classic cell lines (type I) and the remaining classic cell lines (type II). The distinction was based upon both an abrupt 6-fold increase of y-enolase activity and an 18-fold increase of NSE-ag, which for the larger part was enzymatically inactive. Within each group the increase of NSE-ag content was significantly correlated with the increase of y-enolase activity and both NSE-ag content and y-enolase activity increased linearly with Td. It is concluded that y-enolase seems to be associated with the regulation of growth rate and that a compound with the y-enolase antigen but without enzyme activity can distinguish two different classes of SCLC cell lines. Furthermore the demonstration that NSE-ag can represent the active enzyme as well as an enzymatically inactive compound may explain why a controversy about neuron- or non-specificity of NSE exists.
of NSE in
many
Since the beginning of the eighties numerous continuously growing cell-lines from Small Cell Lung Cancer (SCLC) biopsies have been established. Investigators at the National Cancer Institute (Bethesda, USA) were the first to distinguish two types of cell-lines i.e. variant and classic cell-lines, characterised by the absence or presence respectively of the enzyme L-dopadecarboxylase. In comparison with classic cell lines variant cell lines were shown to have a higher growth rate, a higher cloning efficiency, a larger cell volume, a lower content of Neuron-Specific Enolase (NSE), amplification of c-myc, absence of gastrin releasing peptide (GRP) and neurotensin, and a decreased sensitivity to radiotherapy and chemotherapy (Bepler et al., 1987; Carney et al., 1985; Gazdar et al., 1985; Bepler et al., 1989a; Broers et al., 1985; Moody et al., 1985; Broers et al., 1988). Recently Bepler et al. (1989b) added a third class, so-called transitional cells, based on the presence of p64 c-myc in some of the classic cell lines. The addition of a third class of cell lines, is substantiated by intermediate levels of neuroendocrine markers, growth rate and cloning efficiency. NSE is widely used as a neuroendocrine marker. NSEimmuno reactivity is not only seen in neurons, but also in neuroendocrine cells present in endocrine glands and in the diffuse neuroendocrine systems of the lung, intestine, thymus and skin. NSE has been demonstrated in tumours, thought to arise from the neuroendocrine cell system, such as SCLC, neuroblastoma, carcinoid, pancreatic islet cell tumours and medullary thyroid carcinoma (Schmechel et al., 1978; Tapia et al., 1981; Wick et al., 1983). NSE is also present in erythrocytes, lymphocytes and platelets (Marangos et al., 1980b; Hullin et al., 1980) and in malignant lymphomas, testicular cancer, hypernephroma and non-small cell lung cancer (Takashi et al., 1989; Ariyoshi et al., 1983; Kuzmits et al., 1987; Pinto et al., 1989; Oka et al., 1989; Niehans et al. 1988). Such observations question the correlation between NSE and the neuroendocrine cell system (Schmechel, 1985). We have investigated the relationship between NSE-
immunoreactivity and enolase-enzyme activity in SCLC-cell lines. It was found that immunoreactive NSE can represent the active enzyme y-enolase as well as an enzymatically inactive compound. The active enzyme was linearly correlated with the growth rate and the presence of the inactive compound distinguished two different classes of SCLC-cell lines. Materials and methods
SCLC-cell lines The SCLC-cell lines were generously provided by the Dept. of Clinical Immunology, University of Groningen, The Netherlands. The following cell lines were used GLC-1, GLC-2, GLC-3, GLC-4, GLC-1-13, GLC-8, GLC-l 1, GLC14, GLC-16, GLC-19. GLC-28 and GLC-34. The first four cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated foetal calf serum (Gibco), the remaining cell lines in serum free RPMI 1640 supplemented with hydrocortisone, insulin, transferrin, 1 7-p-estradiol, sodium selenite, bombesin and vasopressin as previously described (De Leij et al., 1985).
Sample preparation A cell pellet, containing 1-3 x 106 cells,
haemocytometer.
by
The remaining cells were centrifuged at 800 g and the pellet thawing the cell pellet was suspended in 0.5 ml of 50 mM Tris-HCI buffer pH 8.0 containing 100 mM KCI, 10 mM MgCI2, 2 mM dithiotreitol and 100 mM sucrose. After centrifugation for 10 min at 800 g the supernatant was used for measuring neuron specific enolase was frozen at - 70°C. After
Correspondence: T.A.W. Splinter. Received 26 May 1992; and in revised form 30 July 1992.
was obtained
centrifugation for 10 min at 250g. In order to remove dead cells and cell debris the pellet was washed once with PBS and treated with 1 ml of 0.05% Trypsin-0.02% EDTA (Flow Laboratories) for 3 min at 37°C. To inactivate trypsin culture medium supplemented with 10% foetal calf serum was added. Then DNAse (Sigma DN-25) was added to a final concentration of 0.1%. After mixing an aliquot of the single cell suspension was used for cell counting in a
1066
T.A.W. SPLINTER et al.
immunoreactivity, enolase enzyme activity, enolase isoenzyme composition and protein content. Neuron specific enolase immunoreactivity NSE-immunoreactivity (NSE-ag) was determined with the Pharmacia NSE-RIA, as previously described (Cooper et al., 1985).
Enolase enzyme activity Enolase (2-phospho-D-glycerate hydrolase: EC4.2. 1.11) activity was measured in 100 mM Tris/HCI buffer pH 8.0, containing 100 mM MgCl2, 100 mM KCI, 0.5 mM EDTA, 1.5 mM ADP (Boehringer), 0.2 mM NADH (Sigma), 0.5 U ml-' pyruvate kinase (Boehinger) and 1.0 U ml-' lactate dehydrogenase (Boehringer). The reaction mixture was preincubated for 10 min at 30°C. The reaction was started with the substrate glycero-2-phosphate 1 mM (Boehringer) and assayed kinetically in a Philips PU 8720 spectophotometer at 340 nm and 30°C (Oskam et al., 1985). The supernatant was chosen so as to contain an enolase enzyme activity between 50 and 500 U 1' within these values a linear correlation existed between enolase enzyme activity and protein content of the different cell lines.
Enolase isoenzymes Enolase isoenzymes were separated on cellulose acetate gel (Cellogel) in 20 mM sodium-phosphate buffer pH 7.0 and enolase activity as determined as described previously (Oskram et al., 1985).
L-dopa decarboxylase L-dopa decarboxylase (aromatic L-amino acid decarboxylase; ALAAD) was measured as described previously (Boomsma et al., 1986) and expressed as mU 10-6 cells. For measuring ALAAD activity a frozen cell pellet, containing a known number of cells, was dissolved and further diluted when necessary in bidistilled water containing 40 g I` of bovine serum albumin and 10gl-l of glutathione. Purification of NSE and production of antiserum Human NSE was purified from postmortem human brain cortex and the antiserum was produced in rabbits (Haglid et al., 1973). This antiserum was extensively absorbed with human non-neuronal enolase until the resulting rabbit antiNSE did not show any crossreactivity with a-enolase in an ELISA (Aurell et al., 1989).
Immunoblotting with anti-NSE Samples of the various SCLC-cell lines were sonicated (Branson, Sonifier, cell disruptor B15) in 1% SDS at 90°C until a clear solution was obtained, usually no longer than 60 sec. (Wang et al., 1990). Samples of sonicated SCLC-cell lines were run in SDS gel electrophoresis, using a 5-10% linear polyacrylamide slab gel. The proteins were transferred to a 0.45 ItM nitrocellulose membrance according to Towbin et al. (1979) except that 0.1% SDS was added to the transfer buffer. The electrophoretic blots were detected using rabbit anti-NSE serum (absorbed xix) (diluted 1:500 in TRISbuffered saline) as the first antibody and peroxidaseconjugated goat anti-rabbit IgG (diluted 1:200) as the second antibody Diaminobenzidine (0.5 mg ml-' in TBS) and H202 (0.03%) were used as the enzyme substrate for the colour reaction. Statistical methods The date are presented as the mean ± the standard deviation of the mean. Correlation coefficients were estimated by using a simple linear regression analysis. The significance of the
differences between the means of two groups was tested by the Student's t-test. When a P-value was less than 0.05 the difference was regarded significant. Doubling time (Td) and NSE-immunoreactivity (NSE-ag) During strictly controlled exponential growth the doublingtime, NSE-ag, enolase and ALAAD-enzyme activity were measured in 23 different passages of 11 different cell lines (GLC-1, 2, 4, 1-13, 8, 11, 14, 16, 19, 28 and 34). The first three cell lines (GLC-1, 2 and 4) did not contain ALAAD activity and were therefore called variant, the remaining cell lines were classified as classic. As shown in Figure 1 the NSE-ag/Td ratio distinguished two groups of cell lines: at the left GLC-1, 2, 4, 1-13, 8, 11, 16, 19 and earlier passages of GLC 14 and 28; at the right GLC-34 and later passages of GLC-14 and 28. Each group showed a highly significant correlation between NSE-ag content and Td. (Correlation coefficients were 0.95 and 0.99 of the left and right group respectively, the P-values
'PI
1065-1069 (1992), 66, 1065-1069 (1992), 66,
Macmillan Press Ltd., 1992 1992
Distinction of two different classes of small-cell lung cancer cell lines by enzymatically inactive neuron-specific enolase T.A.W. Splinter', C.F. Verkoelen', M. Vlastuin', T.C. Kok', G. Rijksen2, K.G. Haglid3, F. Boomsma4 & A. van de Gaast' 'Department of Medical Oncology, University Hospital, Dijkzigt, 3015 GD Rotterdam, The Netherlands; 2Department of Haematology, Lab. of Enzymology, University Hospital, 3584 CX Utrecht, The Netherlands; 3Department of Histology, Institute of Neurobiology, University of Goteborg, S-40033 Goteborg, Sweden and 4the Department of Internal Medicine I, University Hospital Dijkzigt 3015 Rotterdam, The Netherlands. Summary Neuron specific enolase (NSE) is widely used
as a neuro-endoctrine marker. However the presence non-neuroendocrine tissues has raised questions on the specificity of NSE. We have investigated NSE immunoreactivity (NSA-ag), y-enolase activity and total enolase activity in small cell lung cancer (SCLC) cell lines. During well-controlled exponential growth comparison of NSE-ag content and y-enolase activity with the doubling-time (Td) and NSE-ag content with y-enolase and total enolase activity led to a clear distinction of two types of cell line: variant cell lines plus part of the classic cell lines (type I) and the remaining classic cell lines (type II). The distinction was based upon both an abrupt 6-fold increase of y-enolase activity and an 18-fold increase of NSE-ag, which for the larger part was enzymatically inactive. Within each group the increase of NSE-ag content was significantly correlated with the increase of y-enolase activity and both NSE-ag content and y-enolase activity increased linearly with Td. It is concluded that y-enolase seems to be associated with the regulation of growth rate and that a compound with the y-enolase antigen but without enzyme activity can distinguish two different classes of SCLC cell lines. Furthermore the demonstration that NSE-ag can represent the active enzyme as well as an enzymatically inactive compound may explain why a controversy about neuron- or non-specificity of NSE exists.
of NSE in
many
Since the beginning of the eighties numerous continuously growing cell-lines from Small Cell Lung Cancer (SCLC) biopsies have been established. Investigators at the National Cancer Institute (Bethesda, USA) were the first to distinguish two types of cell-lines i.e. variant and classic cell-lines, characterised by the absence or presence respectively of the enzyme L-dopadecarboxylase. In comparison with classic cell lines variant cell lines were shown to have a higher growth rate, a higher cloning efficiency, a larger cell volume, a lower content of Neuron-Specific Enolase (NSE), amplification of c-myc, absence of gastrin releasing peptide (GRP) and neurotensin, and a decreased sensitivity to radiotherapy and chemotherapy (Bepler et al., 1987; Carney et al., 1985; Gazdar et al., 1985; Bepler et al., 1989a; Broers et al., 1985; Moody et al., 1985; Broers et al., 1988). Recently Bepler et al. (1989b) added a third class, so-called transitional cells, based on the presence of p64 c-myc in some of the classic cell lines. The addition of a third class of cell lines, is substantiated by intermediate levels of neuroendocrine markers, growth rate and cloning efficiency. NSE is widely used as a neuroendocrine marker. NSEimmuno reactivity is not only seen in neurons, but also in neuroendocrine cells present in endocrine glands and in the diffuse neuroendocrine systems of the lung, intestine, thymus and skin. NSE has been demonstrated in tumours, thought to arise from the neuroendocrine cell system, such as SCLC, neuroblastoma, carcinoid, pancreatic islet cell tumours and medullary thyroid carcinoma (Schmechel et al., 1978; Tapia et al., 1981; Wick et al., 1983). NSE is also present in erythrocytes, lymphocytes and platelets (Marangos et al., 1980b; Hullin et al., 1980) and in malignant lymphomas, testicular cancer, hypernephroma and non-small cell lung cancer (Takashi et al., 1989; Ariyoshi et al., 1983; Kuzmits et al., 1987; Pinto et al., 1989; Oka et al., 1989; Niehans et al. 1988). Such observations question the correlation between NSE and the neuroendocrine cell system (Schmechel, 1985). We have investigated the relationship between NSE-
immunoreactivity and enolase-enzyme activity in SCLC-cell lines. It was found that immunoreactive NSE can represent the active enzyme y-enolase as well as an enzymatically inactive compound. The active enzyme was linearly correlated with the growth rate and the presence of the inactive compound distinguished two different classes of SCLC-cell lines. Materials and methods
SCLC-cell lines The SCLC-cell lines were generously provided by the Dept. of Clinical Immunology, University of Groningen, The Netherlands. The following cell lines were used GLC-1, GLC-2, GLC-3, GLC-4, GLC-1-13, GLC-8, GLC-l 1, GLC14, GLC-16, GLC-19. GLC-28 and GLC-34. The first four cell lines were cultured in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated foetal calf serum (Gibco), the remaining cell lines in serum free RPMI 1640 supplemented with hydrocortisone, insulin, transferrin, 1 7-p-estradiol, sodium selenite, bombesin and vasopressin as previously described (De Leij et al., 1985).
Sample preparation A cell pellet, containing 1-3 x 106 cells,
haemocytometer.
by
The remaining cells were centrifuged at 800 g and the pellet thawing the cell pellet was suspended in 0.5 ml of 50 mM Tris-HCI buffer pH 8.0 containing 100 mM KCI, 10 mM MgCI2, 2 mM dithiotreitol and 100 mM sucrose. After centrifugation for 10 min at 800 g the supernatant was used for measuring neuron specific enolase was frozen at - 70°C. After
Correspondence: T.A.W. Splinter. Received 26 May 1992; and in revised form 30 July 1992.
was obtained
centrifugation for 10 min at 250g. In order to remove dead cells and cell debris the pellet was washed once with PBS and treated with 1 ml of 0.05% Trypsin-0.02% EDTA (Flow Laboratories) for 3 min at 37°C. To inactivate trypsin culture medium supplemented with 10% foetal calf serum was added. Then DNAse (Sigma DN-25) was added to a final concentration of 0.1%. After mixing an aliquot of the single cell suspension was used for cell counting in a
1066
T.A.W. SPLINTER et al.
immunoreactivity, enolase enzyme activity, enolase isoenzyme composition and protein content. Neuron specific enolase immunoreactivity NSE-immunoreactivity (NSE-ag) was determined with the Pharmacia NSE-RIA, as previously described (Cooper et al., 1985).
Enolase enzyme activity Enolase (2-phospho-D-glycerate hydrolase: EC4.2. 1.11) activity was measured in 100 mM Tris/HCI buffer pH 8.0, containing 100 mM MgCl2, 100 mM KCI, 0.5 mM EDTA, 1.5 mM ADP (Boehringer), 0.2 mM NADH (Sigma), 0.5 U ml-' pyruvate kinase (Boehinger) and 1.0 U ml-' lactate dehydrogenase (Boehringer). The reaction mixture was preincubated for 10 min at 30°C. The reaction was started with the substrate glycero-2-phosphate 1 mM (Boehringer) and assayed kinetically in a Philips PU 8720 spectophotometer at 340 nm and 30°C (Oskam et al., 1985). The supernatant was chosen so as to contain an enolase enzyme activity between 50 and 500 U 1' within these values a linear correlation existed between enolase enzyme activity and protein content of the different cell lines.
Enolase isoenzymes Enolase isoenzymes were separated on cellulose acetate gel (Cellogel) in 20 mM sodium-phosphate buffer pH 7.0 and enolase activity as determined as described previously (Oskram et al., 1985).
L-dopa decarboxylase L-dopa decarboxylase (aromatic L-amino acid decarboxylase; ALAAD) was measured as described previously (Boomsma et al., 1986) and expressed as mU 10-6 cells. For measuring ALAAD activity a frozen cell pellet, containing a known number of cells, was dissolved and further diluted when necessary in bidistilled water containing 40 g I` of bovine serum albumin and 10gl-l of glutathione. Purification of NSE and production of antiserum Human NSE was purified from postmortem human brain cortex and the antiserum was produced in rabbits (Haglid et al., 1973). This antiserum was extensively absorbed with human non-neuronal enolase until the resulting rabbit antiNSE did not show any crossreactivity with a-enolase in an ELISA (Aurell et al., 1989).
Immunoblotting with anti-NSE Samples of the various SCLC-cell lines were sonicated (Branson, Sonifier, cell disruptor B15) in 1% SDS at 90°C until a clear solution was obtained, usually no longer than 60 sec. (Wang et al., 1990). Samples of sonicated SCLC-cell lines were run in SDS gel electrophoresis, using a 5-10% linear polyacrylamide slab gel. The proteins were transferred to a 0.45 ItM nitrocellulose membrance according to Towbin et al. (1979) except that 0.1% SDS was added to the transfer buffer. The electrophoretic blots were detected using rabbit anti-NSE serum (absorbed xix) (diluted 1:500 in TRISbuffered saline) as the first antibody and peroxidaseconjugated goat anti-rabbit IgG (diluted 1:200) as the second antibody Diaminobenzidine (0.5 mg ml-' in TBS) and H202 (0.03%) were used as the enzyme substrate for the colour reaction. Statistical methods The date are presented as the mean ± the standard deviation of the mean. Correlation coefficients were estimated by using a simple linear regression analysis. The significance of the
differences between the means of two groups was tested by the Student's t-test. When a P-value was less than 0.05 the difference was regarded significant. Doubling time (Td) and NSE-immunoreactivity (NSE-ag) During strictly controlled exponential growth the doublingtime, NSE-ag, enolase and ALAAD-enzyme activity were measured in 23 different passages of 11 different cell lines (GLC-1, 2, 4, 1-13, 8, 11, 14, 16, 19, 28 and 34). The first three cell lines (GLC-1, 2 and 4) did not contain ALAAD activity and were therefore called variant, the remaining cell lines were classified as classic. As shown in Figure 1 the NSE-ag/Td ratio distinguished two groups of cell lines: at the left GLC-1, 2, 4, 1-13, 8, 11, 16, 19 and earlier passages of GLC 14 and 28; at the right GLC-34 and later passages of GLC-14 and 28. Each group showed a highly significant correlation between NSE-ag content and Td. (Correlation coefficients were 0.95 and 0.99 of the left and right group respectively, the P-values
